23 research outputs found
Genipin-enhanced fibrin hydrogel combined with engineered silk composite for intervertebral disc repair
BMP Antagonists - A Possible Cause for Spinal Non-Fusion?
INTRODUCTION:Spinal fusion is a procedure where the intervertebral disc (IVD) is removed and two adjacent vertebrae are forced to fuse by compression. This procedure is the most commonly applied procedure to achieve spinal stability and relief of back pain. However, non-successful fusion leads to pseudo-athrosis and ongoing pain. There is increasing evidence that supraphysiological doses of BMP2 and burst-release of this cytokine did not generate satisfying results in clinical studies. Current hypothesis was raised that IVD cells and/or tissue seem to inhibit the action of BMP2. In this overview we summarize the current evidence that BMPs might be inhibited by the secretome of human IVD cells, i.e., nucleus pulposus cells (NPC), annulus fibrosus cells (AFC) and cartilaginous endplate (CEPC) cells.
METHODS:We stimulated low-passage (2-3) human bonemarrow-derived mesenchymal stromal cells (MSCs) and femoral hip-derived osteoblasts (OBs) and co-cultured these with allogeneic IVD cells obtained from spinal surgery. We then stimulated MSCs and the OBs in monolayer and osteogenic medium, whereas IVD cells were kept in 3D alginate bead culture and separated by high density pore culture inserts (0.4 µm pore size). We quantified relative gene expression at bone-relevant genes, alkaline phosphatase (ALP) activity and Alizarin red (ALZR) staining after 21 days. Furthermore, to test the effect of a previously investigated BMP2 analog to block the inhibitors, cells were further stimulated with 100 ng/mL BMP2 and/or L51P.
RESULTS:We found significant inhibitory effects of IVD cells onto MSCs undergoing differentiation in presence of NPC, AFC and CEPC as shown in reduced osteogenic gene expression, ALZR staining and ALP activity (N = 11 donors paired on each side). In the case of allogeneic human OBs only a trend towards inhibition could be demonstrated (N = 7 donors on each side). The addition of L51P to the coculture recovered ossification. On the side of the IVD cells BMP2 and/or L51P had a strong chondrogenic effect.
DISCUSSION & CONCLUSIONS:Our data suggested evidence for inhibition for MSCs. However, OBs did not show the same inhibitory effects but showed a trend in presence of IVD’s secretome. This warrants for animal models where the donor variance can be better controlled.
ACKNOWLEDGEMENTS:This work was supported by a start-up grant from the Center for Applied Biotechnology and Molecular Medicine (CABMM). Further funds were received from the Swiss Society of Orthopaedics (SGOT), the clinical trials unit (CTU) of Bern University Hospital, and by a Eurospine Task Force Research grant #2019_22
The regenerative effects of two common platelet-rich plasma production methods on human anterior cruciate ligamentocytes
Comparing two common production methods for platelet-rich plasma and the boosting effect for human anterior cruciate ligamentocytes
Efficient Nonviral Transfection of Primary Intervertebral Disc Cells by Electroporation for Tissue Engineering Application.
Low back pain (LBP) is an increasing global health problem associated with intervertebral disc (IVD) trauma and degeneration. Current treatment options include surgical interventions with partial unsatisfactory outcomes reported such as failure to relieve LBP, nonunions, nerve injuries, or adjacent segment disease. Cell-based therapy and tissue engineered IVD constructs supplemented with transfected disc cells that incorporate factors enhancing matrix synthesis represent an appealing approach to regenerate the IVD. Gene delivery approaches using transient nonviral gene therapy by electroporation are of a high clinical translational value since the incorporated DNA is lost after few cell generations, leaving the host's genome unmodified. Human primary cells isolated from clinically relevant samples were generally found very hard to transfect compared to cell lines. In this study, we present a range of parameters (voltage pulse, number, and duration) from the Neon(®) Transfection System for efficient transfection of human and bovine IVD cells. To demonstrate efficiency, these primary cells were exemplarily transfected with the commercially available plasmid pCMV6-AC-GFP tagged with copepod turbo green fluorescent protein. Flow cytometry was subsequently applied to quantify transfection efficiency. Our results showed that two pulses of 1400 V for 20 ms revealed good and reproducible results for both human and bovine IVD cells with efficiencies ≥47%. The presented parameters allow for successful human and bovine IVD cell transfection and provide an opportunity for subsequent regenerative medicine application
Stimulation of intervertebral disc cells in alginate bead culture with bone morphogenetic protein 2 and/or L51P
INTRODUCTION: In clinics, Bone Morphogenetic Protein 2 (BMP2) was applied for the support during spinal fusion. Further BMP2 was tested in IVD models and showed potential for IVD regeneration. The aim of this study is the investigation of BMP2 and the BMP2 analogue, L51P, on different cell types of the human IVD in 3D alginate beads, particularly their plasticity to undergo bone formation.
METHODS: Human nucleus pulposus (NPC), annulus fibrosus (AFC) and cartilaginous endplate cells (CEPC) were each encapsulated in 1.2% alginate at a density of 4 Mio/mL. NPC, AFC, and CEPC beads were then cultured in α-MEM or osteogenic medium (OM) supplemented with 10% FBS and 100 ng/mL BMP2 and/or L51P for 21 days. Medium supplemented with cytokines was refreshed twice per week. Beads were snap frozen with liquid nitrogen after 7 days for mRNA analysis of Aggrecan (ACAN), Collagen type1 (COL1), Collagen type 2 (COL2) and runt-related transcription factor 2 (RUNX2) qPCR. Further, beads were stained with Alcian Blue after 21 days.
RESULTS & DISCUSSION: ACAN expression was the highest up-regulated in IVD cells stimulated with OM and 100 ng/mL BMP2 and L51P compared to the negative control (basal medium only) in NPC, AFC and CEPC (mean ± SEM NP: 18.95 ± 15.65). The same was true for COL2 expression (NP: 72.47 ± 62.95). COL1 remained unaffected (N=2).
CONCLUSIONS: In this study, we showed the trend of an increase in ACAN and COL2 gene expression in stimulated cells. Interestingly the co-treatment of BMP2 and L51P showed a cumulative effect towards an increased ECM production